Biological Tests Results

Biological Tests Results

MRET ACTIVATED WATER AND ITS SUCCESFUL APPLICATION FOR PREVENTIVE TREATMENT AND ENHANCED TUMOR RESISTANCE IN ONCOLOGY

The goal of this investigation was to study on mice the effect of MRET activated water as a potential agent for the prevention and treatment of two kinds of oncology diseases (laboratory models of Ehrlich's ascites tumor and Sarcoma ascites form). Significant positive effect of MRET activated water regarding the tumor resistance on animals was observed in the experiments conducted on 500 mice (22 groups with 20 mice in each group and 10 groups with 5 mice in each group). The best results were observed in the groups of mice on MRET water activated for 30 minutes (optimal regime of activation). The substantial anti-tumor efficacy was confirmed by very high level of reduction of Total Number of Viable Tumor Cells by 76%in “preventive treatment” and by 55% in “therapeutic treatment” regime. The life span of mice which received optimal activated water increased by 61%in “preventive treatment” and by 43% in “therapeutic treatment” regime. The significant anti-tumor effect of MRET Activated Water on mice was close to the action of the chemotherapy agents and allowed to avoid the side effects that typically follow chemotherapy treatment in oncology.

Fig 1: The effect of preventive (1–5) and therapeutic (6–10) application of MRET activated water on average total number of viable cells <C> in an ascetic tumor, obtained from mice inoculated intraperitoneally with tumor cells of Ehrlich carcinoma.

Fig 2: The change of the percentage increase of life span of tumor-bearing mice with ascitic Ehrlich carcinoma which received different types of MRET activated water in “preventive treatment” and “therapeutic treatment” regimes. The digits under the charts correspond with the duration of water activation in minutes.

Fig 3: The effect of MRET activated water on cytotoxic activity of lymphocytes containing NK-cells. Activated water was applied for mice without tumors in two regimes (for 21 and 14 days), called “preventive” (1–5) and “therapeutic” (6–10).

Pic: The appearance of mice from “control” (A) and “preventive treatment” groups (optimal activation time 30 minutes) (B) on the 18th day after ascitic Ehrlich carcinoma cell inoculation.

The results of investigation of the application of water activated by non-ionizing Molecular Resonance Effect Technology (MRET) process for prevention treatment and enhancement of tumor resistance of animals to two types of oncology diseases in vivo on 500 mice are presented. The research conducted on physical parameters of water confirmed that MRET activation process contributed to substantial modification of the basic physical-molecular properties of distilled water (substantial reduction of viscosity as a function of applied tangent pressure, as well as substantial decrease of electrical conductivity and dielectric permittivity as functions of the frequencies of applied electromagnetic field). The significant positive effect of MRET activated water on tumor resistance of animals was observed in the process of this investigation in all groups of mice on different fractions of activated water. The best results were observed in the groups of mice on MRET water activated for 30 minutes (optimal regime of activation). The results were better in “preventive treatment” regime compare to “therapeutic treatment” regime. Additionally, this investigation confirmed that the long-term preservation of activated water at low temperature (around 0°C) for 45 days decreased its anti-tumor efficacy but left it on the significantly high level compare to other fractions. The test results point at the dual mechanism of MRET water effect on tumors: the prevention and inhibition of tumor growth together with the reduction of quantity of viable tumor cells. The significant anti-tumor effect of MRET activated distilled water on mice was close to the action of the chemotherapy agents and allowed to avoid the side effects that typically follow chemotherapy treatment of oncology.

In the process of investigation of cytotoxic activity of NK-cells the significant increase of lymphocyte cytotoxicity levels was observed when donor mice were treated with MRET water activated for 30 minutes. The results also showed that the extension of the application of MRET water from 14 days to 21 days significantly increased the value of cytotoxicity index. It is possible to admit that the extension of time of application of MRET water will lead to higher levels of enhancement in NK-cells activity. Thus, the application of MRET activated water can be quite promising approach for non-drug stimulation of NK-cells immunization vaccines.

The detailed description of this investigation is presented in “BENEFITS MRET WATER”.

The in vitro investigations on normal PMBC cells (peripheral blood mononuclear cell) and on HeLa cancer cells (cell line ATCC # CCL-2 cervical adenocarcinoma) and were conducted under the supervision of Patrick Pezzoli, Ph.D. at AltheaDx Technologies, San Diego, USA. The experiments analyzed: cells lysed at 0 hour, cells cultured for 24 hours in untreated medium and cells cultured for 24 hours in medium treated with MRET activator for 30 minutes. DNA samples from each batch were processed and the resultant data was analyzed using Affymetrix Genotyping Console 3.0 to obtain genotype calls and copy number calls. Cell counts and % viability were obtained using the Trypan Blue exclusion technique.

The experimental data revealed no difference between the zero hour (control), MRET treated and untreated samples in term of genotypes and copy number calls. Thus, MRET activation of water based medium did not induce any changes in cells on genetic level.

The studies showed that in MRET activated medium the viability of normal cells (PBMC) was higher (Table 1), and the viability of cancer cells (HeLa) was lower (Table 2) compared to their viability in untreated medium.

Table 1: PBMC cell counts and % viability

Sample

Cell Count

% Viability

Viable cells

0 hour

3.27x106

91.5

2.99x106

Untreated

8.70x105

83.4

0.73x106

Treated

7.73x105

88.8

0.69x106

Table 2: HeLa cell counts and % viability

Sample

Cell Count

% Viability

Viable cells

0 hour

3x106

92

3.27x106

Untreated

7x106

97

6.79x106

Treated

5x106

92

4.60x106

For normal cells (PBMC) the changes in cell counts were similar for untreated and MRET treated medium (Fig 1). Thus, MRET treatment did not affect the growth of normal cells.

For cancer cells (HeLa) the experimental data revealed significant inhibition of cancer cells growth in MRET treated medium. The growth of viable cancer cells was inhibited by 54% in MRET treated medium compared to untreated medium (Fig 2).Fig 1: Viable PBMC cell counts after 24 hours of incubation Fig 2: Viable HeLa cell counts after 24 hours of incubation
The results of AltheaDx research on HeLa cancer cells in vitro support the results obtained earlier in the investigation regarding the effects of MRET water on tumor resistance in animal model. The study on 500 mice was conducted under the supervision of Professor V. Vysotskii, S. Olishevsky, Ph.D. and Y. Yanish, Ph.D. at Kyiv Institute of Experimental Pathology, Oncology and Radiobiology of Ukrainian Academy of Science. It showed substantial inhibition of growth of viable tumor cells following the consumption of MRET water. In the course of this investigation the groups of mice in “preventive regime” ingested MRET water for 2 weeks prior to the inoculation of Ehrlich carcinoma cancer cells and for 3 weeks after the inoculation. The groups of mice in “therapeutic regime” ingested MRET water only during 3 weeks after the inoculation of Ehrlich carcinoma cancer cells. Following the consumption of MRET water activated for 30 minutes (the optimal time of activation) the growth of viable tumor cells was inhibited by 76% in “preventive regime” and by 55% in “therapeutic regime.”

As a conclusion it is possible to say that the studies conducted at AltheaDx Technology confirmed that MRET activated water did not affect the cells on genetic level; it affected the morphology of normal cells in positive way increasing their viability and promoted significant inhibition of growth of cancer cells.

A clinical observation was conducted on a patient undergoing chemotherapy treatment for metastasized naso-pharyngeal cancer. On a regular basis, WBC counts usually decrease to very low level. The rebounding of WBC from this low range takes about 3-5 weeks. The ingestion of MRET water prevented the decrease of WBC counts to their lowest level and helped to regain the pre-chemotherapy level within unusually short period of time of 3 days.

Thus, the clinical observation revealed that on the 10th day after the beginning of chemotherapy (in 3 days after WBC counts dropped to 10% of pre-chemotherapy level) WBC counts rebounded to 93% of their pre-chemotherapy level. In compliance with Student’s Distribution criteria the rebounding of WBC was higher than 70% of pre-chemotherapy level with p=0.05.

Testing conducted at C.A.I. Environmental Laboratory, Carlsbad, USA revealed the significant reduction of the amount of total coliforms following the process of MRET activation. In the rainwater activated for 30 minutes the amount of total coliforms decreased by 86% after the process of water activation. This test confirms that MRET water acts as antibacterial agent, providing sterilization effect.

The investigation revealed the significant effect of MRET-activated nutrient medium in aerobic environment on the process of growth and reproduction of E.coli microorganisms, their division, the size of colonies and the modification of forms of culture cells. It was observed that at low initial concentration of cells of investigated culture Escherichia coli K-12 MRET nutrient medium activated during 30 minutes and 60 minutes inhibited the growth of culture 27 and 303 times respectively during the 25 hours of experiment.

Initial view of Petri dishes with different fractions of nutrient medium at the beginning of experiment is presented on Fig 1.

Fig 1: Petri dishes at the beginning of experiment. Identical very small amount of Escherichia coli K-12 cells was introduced on a surface of non-activated nutrient medium of control dishes and on a surface of dishes with nutrient medium MRET-activated for 30 and 60 minutes(tact=0.5h and tact=1.0h) in aerobic environment. There are no colonies of microorganisms in Petri dishes in the beginning of experiment.

Petri dishes with grown colonies and statistical parameters of the colonies after 23 hours of experiments are presented on Fig 2: (a) non-activated nutrient medium (control); (b) nutrient medium activated for 30 minutes; (c) nutrient medium activated for 60 minutes. Petri dishes after 29 hours of experiment are shown on Fig 3. The significant inhibition of growth of E.coli in activated samples was revealed and it confirmed the strong bacteriostatic effect of MRET-activated medium.

This experiment shows that MRET-activation process has very strong bacteriostatic effect on conditionally pathogenic E.coli microorganisms and that the inhibition of E.coli growth is more effective when activation time is increased. It was observed that at low initial concentration of cells of E.coli in nutrient medium MRET-activation during 30 minutes and 60 minutes period of time inhibited the culture growth NC/N0.5 = 27 and NC/N1.0 = 303 times respectively after 25 hours of experiment (Fig 4). Consequently, the level of bacteriostatic activity was 96% in 30 minutes activated nutrient medium and 99.7% in 60 minutes activated medium. Thus, the direct correlation between bacteriostatic activity of MRET-activated nutrient medium and the time of activation was confirmed.

This experiment also revealed the strong effect of MRET-activated water on the process of division of E.coli microorganisms, the modification of forms of culture cells and the size of colonies. It was observed that one of the reasons of abnormally low growth of E.coli bacteria was related to the modification of the process of cell division in MRET-activated nutrient medium. In the process of growth and reproduction a large number of cells did not separate from each other and the linear line-ups consisting of 2-3 sequentially paired cells were formed. The culture cells grown in non-activated and MRET activated medium are shown on Fig 5 and Fig 6 respectively.

II. The effect of MRET water on metabolic activity of E.coli bacteria in aerobic and anaerobic environment

Reductant activity is an integral characteristic of metabolic activity of microorganisms and it is measured with the help of Sodium Resasurine color indicator in the percentage degree of discoloration (purple = 0%, red = 50%, transparent = 100%). The reductant activity of E.coli bacteria reduced up to 3 times in 30 minutes MRET-activated water and up to 1.6 times in 60 minutes activated water during the first 6 hours of experiment in aerobic environment (Fig 7 and 8).

This experiment showed that there was no direct correlation between the inhibition of metabolic (reductant) activity and the inhibition of growth of E.coli (bacteriostatic activity) in MRET activated water. The bacteriostatic effect is substantially higher in 60 minutes activated water and the inhibition of reductant activity during the first 3 hours is higher in 30 minutes activated water. Thus, this experiment revealed that the optimum time of activation for the maximum inhibition of metabolic activity of E.coli bacteria in aerobic environment is 30 minutes. The same optimum time of activation was found in the process of another investigation regarding the application of MRET activated water for preventive treatment and enhancement of tumor resistance in vivo on 500 mice for two types of cancer conducted at Kiev Institute of Experimental Pathology, Oncology and Radiobiology of Ukrainian Academy of Science.

Taking in consideration that a small population of pathogenic bacteria, such as E.coli, is usually presented in complex microbial associations in the intestine of the body, the test on metabolic activity of E.coli bacteria in anaerobic environment was conducted. Anaerobic environment simulates the environmental conditions similar to the conditions in the intestine of humans and animals. The investigation showed that the reductant activity of E.coli bacteria in anaerobic environment practically did not change (Fig 9 and 10).

This experiment revealed that the process of MRET activation did not have any significant effect on reductant activity of E.coli bacteria in anaerobic environment.

III. The stimulating effect of MRET water on metabolic activity of Complex Microbial Associations in anaerobic environment

In order to simulate the environmental conditions similar to the conditions in the intestine of humans and animals the test on metabolic activity of microbial associations was conducted in anaerobic environment. It was found that MRET-activated water substantially increased reductant activity of complex microbial associations during the first several hours of experiment (Fig 11).

This experiment revealed that the optimum time of activation for the maximum increase of metabolic activity of microbial associations in anaerobic environment was 30 minutes. The same optimum time of activation was found in the process of inhibition of metabolic activity of E.coli in aerobic environment and in another investigation regarding the application of MRET activated water for preventive treatment and enhancement of tumor resistance in vivo.

CONCLUSIONS

This investigation revealed that at low initial concentration of cells of conditionally pathogenic microbiological culture Escherichia coli K-12 in water based nutrient medium activated for 30 minutes and 60 minutes the growth of culture was inhibited 27 and 303 times respectively after the 25 hours of experiment in aerobic environment. This experiment also revealed the strong effect of MRET activated water on the process of division of E.coli microorganisms, the modification of forms of culture cells and the size of colonies. It was observed that one of the reasons of abnormally low growth of E.coli population was related to the modification of the process of cell division in MRET-activated nutrient medium. These results allow admitting that the process of MRET activation and the sterilization effect of MRET water can be applied in food industry and for water purification.

The second stage of investigation revealed that the metabolic (reductant) activity of E.coli bacteria reduced up to 3 times in 30 minutes activated water and up to 1.6 times in 60 minutes activated water during the first 6 hours of experiment in aerobic environment. Another experiment showed that the process of MRET-activation did not affect the reductant activity of E.coli bacteria in anaerobic environment and, consequently, should not affect a small population of conditionally pathogenic bacteria, such as E.coli, usually presented in microbial associations in the intestine of the body.

In order to simulate the environmental conditions similar to the conditions in the intestine of humans and animals the test on metabolic activity of complex microbial associations was conducted in anaerobic environment. It was discovered that MRET activated process substantially increased reductant activity of complex microbial associations during the first hours of experiment. The same 30 minutes optimum time of activation was observed in the process of inhibition of metabolic activity of conditionally pathogenic E.coli bacteria in aerobic environment and for the maximum increase of metabolic activity of complex microbial associations in anaerobic environment (presented in the intestine). The previous investigation regarding the application of MRET activated water for preventive treatment and enhancement of tumor resistance in oncology in vivo on 500 mice also showed the best results on 30 minutes MRET-activated water. Thus, this investigation shows that the ingestion of MRET water is beneficial for the process of digestion and can enhance metabolism of the body.

The Effect of MRET Activated Water on Staphylococcal Infection in vivo in Animal Model (on the cells of immune system) and in vitro on the Culture of Staphylococcus aureus Wood-46

I. The Effect of MRET Water on Staphylococcal Infection in vivo in Animal Model

The research was conducted at Kiyv National Shevchenko University, Ukraine on 400 mice. The mice-male of line BALB in the age of 11 – 13 weeks and of the weight 18 – 21 grams were used. In the process of investigation all mice were divided in three groups. Prior to the inoculation of Staphylococcus aureus Wood-46 culture one group of mice consumed MRET water during 4 weeks (Group 1), another group consumed MRET water during 2 weeks (Group 2), the control group consumed non-activated ordinary distilled water. During the following 2 weeks of experiment the first two groups continued to consume MRET water and the control group consumed ordinary distilled water. The first line of experiments was conducted on 225 mice in order to analyze the persistence of pathogen in homogenate of kidneys of mice comparing 5 groups of mice (two Group 1 and two Group 2 on 15 minutes and 30 minutes MRET activated water and Control). After preliminary experiments the optimal 30 minutes MRET activated water (distilled) was chosen for the main line of investigation.

RESULTS:

1. Significant protective properties of MRET water.

The significant protective properties of MRET water were confirmed by substantial decrease of Staphylococcus CFU (colony forming units) in homogenate of kidneys of mice on MRET water compare to control group of mice following the intra-peritoneal staphylococcal infection after the first 24 hours. The analysis of data in the beginning of experiments leads to the conclusion that significant decrease of pathogen’s colonies in homogenate of kidneys of mice on MRET water begins only after 24 hours following the inoculation of Staphylococcus culture. The results on 30 minutes activated water were much better than on 15 minutes activated water and all further experiments were conducted on 30 minute activated water.

2. The consumption of MRET water eliminated the death rate from 30% (control group) to 0% (MRET group) during the first 9 days of experiment.

There was no case of animal death in all investigated groups within the first 24 hours after intra-peritoneal inoculation of Staphylococcus culture, which is a pretty standard result. During the next 8 days 30% of animals died in control group which also is a standard result. But there were no death cases in both groups of mice that ingested MRET activated water and it is a remarkable result. Nevertheless, the main consequences of Staphylococcus infection do not manifest in death of animals as in case of oncology diseases. Staphylococcus virus affects the live systems and organs of the body. These pathogenic microorganisms cause inflammations, suppurations, abscesses, furuncles, quinsy, cepsical conditions, etc. That’s why a detailed investigation of the process of stimulation by MRET water of phagocytes and of lymphoid organs of immune system of mice infected with Staphylococcus aureus culture was conducted and is presented in this report.

3. The development of the local acute inflammation is essentially suppressed in case of ingestion of MRET activated water.

The local inflammation was induced with the help of the inoculation of Staphylococcus aureus culture into the hinder left paw. The ordinary inflammatory reaction was observed in the group of mice on non-activated water: the intensive reddening of the hinder left paw (Fig 1). Both groups of mice on MRET water did not develop any reddening the hinder left paw inoculated with Staphylococcus aureus culture (Fig 2). The results of this experiment confirm the fact of the substantial inhibition of inflammatory infection in case of the regular consumption of MRET water.

Fig 1: The view of paws of a mouse on non-activated water (reddenning of the injection paw) in 24 hours after the injection of Staphylococcus culture.

Fig 2: The view of paws of a mouse on MRET activated water (no reddenning of the injection paw) in 24 hours after the injection of Staphylococcus culture.

4. The consumption of MRET water stimulates the activity of phagocytic system and the level of natural resistance of animals to pathogenic microorganisms.

For the following series of experiments the inoculation of Staphylococcus aureus Wood-46 was conducted intra-peritoneal in dose LD30 in order to spread infection all over the body.

The phagocytic system is one of the main factors of natural non-specific cellular resistance to infections and inflammations. It is the first line of protection of an organism against penetration and reproduction of pathogenic microorganisms. The protective role of phagocytic cells is based on their capacity to identify, engulf and utilize the alien agents penetrated into internal environment of a macro-organism. Phagocytosis is the main mechanism of natural resistance especially at the first stage of contagious process; it is a regular part of formation of the specific immune response.

The most common methodology applied in the studies of the functional activity of phagocytes is the examination of their phagocytic (engulfing of alien cells) and oxygen-dependent bactericidal activity. Phagocytic activity of neutrophils and macrophages is estimated based on Index of Phagocytosis (percentage of the phagocytes which engulfed test-bacteria) and on Phagocytic Number (average number of test-bacteria engulfed by one phagocyte). The cultures of Staphylococcus aureus and Latex are usually used as test-bacteria. The oxygen-dependent bactericidal activity of phagocytes is studied with the help of NBT-test: an oxygen-dependent reduction of Nitro Blue Tetrazolium into an insoluble Diformazan of Nitro Blue Tetrazolium derivative by phagocytes. With the help of NBT-test it is possible to distinguish the activated phagocytes from the non-activated ones.

MRET water stimulated the phagocytic capacities of neutrophils of a peripheral blood and peritoneal macrophages increasing their phagocytic activity, particularly Phagocytic Indexs (Fig 3A) and Phagocytic Number (Fig 3B). It also stimulated their oxygen-dependent bactericidal activity, particularly the increase of quantity of NBT-positive phagocytes (Fig 4).

Fig 3A: Phagocytic Index of neutrophils and macrophages in two weeks of experiment (object of phagocytosis - Staphylococcus aureus): 1 – Control group; 2 – Mice on MRET water (preventive for 4 weeks); 3 – Mice on MRET water (preventive for 2 weeks).

Fig 3B: Phagocytic Number of neutrophils and macrophags in two weeks of experiment (object of phagocytosis – Staphylococcus aureus): 1 – Control group; 2 – Mice on MRET water (preventive for 4 weeks); 3 – Mice on MRET water (preventive for 2 weeks).

Fig 4: Oxygen-depended bactericidal activity (NBT-test) of neutrophils and macrophages in two weeks of experiment:
1 – Control group; 2 – Mice on MRET water (preventive for 4 weeks); 3 – Mice on MRET water (preventive for 2 weeks).

These experiments confirmed the increase of effective potentials of phagocytes which constitute one of the main factors of natural protection of an organism and initiate the immune response.

The analysis of data in the beginning of experiments leads to the conclusion that significant intensification of phagocytic and bactericidal activity of macrophages and neutraphils of mice on MRET water begins only after 24 hours following the intra-peritoneal inoculation of Staphylococcus culture. At the end of two weeks of experiment the mean values of studied parameters in both groups of mice on MRET water substantially increased compare to the control group. The differences in mean values of the parameters of functional activity of phagocytes of groups of mice consuming MRET water compare to the control group of mice on non-activated water were statistically significant with p<0.05 (for Phagocytic Index and NBT-test). These results confirm the significant intensification of phagocytic and bactericidal activity and of immune system response following the consumption of MRET water.

The differences in mean values of studied parameters for the groups of mice on MRET water compare to each other were statistically insignificant, which confirms the similarity of the level of beneficial effect of MRET water in both groups. This fact also confirms that the regular consumption of MRET water provides health benefits in rather short time (2 weeks in case of the animal mice model).

5. The consumption of MRET water substantially enhances the immune activity of lymphoid organs.

By the end of another series of experiments in both groups of mice on MRET water was observed substantial statistically significant (p<0.05) increase of the weight and the cellularity (quantity of cells) of a spleen and lymph nodes as well as the insignificant increase of weight and cellularity of thymus (Fig 5 and Fig 6).

Fig 5: The weight of lymphoid organs in two weeks of experiment: 1 – Control group; 2 – Mice on MRET water (preventive for 4 weeks); 3 – Mice on MRET water (preventive for 2 weeks).

Fig 6: The cellularity of lymphoid organs in two weeks of experiment: 1 – Control group; 2 – Mice on MRET water (preventive for 4 weeks); 3 – Mice on MRET water (preventive for 2 weeks).

These results confirm the fact of significant intensification of immune system response in animals on MRET water subject to Staphylococcus infection. The difference in studied parameters between the groups of mice on MRET water (4 weeks and 2 weeks of preventive consumption of MRET water) was insignificant which confirms quite fast beneficial effect of MRET water on the immune activity of lymphoid organs.

In the beginning of experiment the cellularity and the weight of lymphoid organs in MRET groups did not show the distinct tendency to modifications. It is reasonable to admit that the consumption of MRET water affects the weight and the cellularity of lymphoid organs only during the infection period.

CONCLUSIONS:

The consumption of MRET activated water significantly enhances the factors of natural resistance of the body which constitute the first line of protection of an organism against the penetration and reproduction of pathogenic microorganisms.

The analysis of data in the beginning of experiment leads to the conclusion that significant changes in all studied parameters of mice on MRET water (decrease of pathogen colonies in homogenate of kidneys, increase of the weight and the cellularity of lymphoid organs, intensification of the phagocytic and bactericidal activity of macrophages and neutraphils) begins only after 24 hours following the inoculation of Staphylococcus culture. Another words the consumption of MRET water increases the potentials of immune capacities of the body to counteract the infections without any changes in the vital parameters of immune organs and functions prior to the penetration of infectious pathogens in the body.

At the end of two weeks of experiment the mean values of studied parameters in both groups of mice on MRET water (preventive for 4 and 2 weeks respectively) significantly increased compare to the control group. The differences in mean values of the studied parameters of the groups of mice consuming MRET water compare to the control group of mice on non-activated water were statistically significant with p<0.05 (for most of the parameters). These results confirm the significant intensification of phagocytic activity and of immune system response following the consumption of MRET water.

The differences in mean values of studied parameters for the groups of mice on MRET water compare to each other were statistically insignificant, which confirms the similarity of the level of the beneficial effect of MRET water in both groups. This fact also confirms that the regular consumption of MRET water provides health benefits in rather short period of time (2 weeks in case of the animal mice model).

II. The Effect of MRET Activation on the Process of Growth of Staphylococcal Culture in Nutrient Medium

The following experiments were designed to study the effect of MRET activation on the process of growth and development of Staphylococcus aureus Wood-46 culture in vitro in nutrient medium (MPA – meat-peptone agar). The bacterial culture was grown overnight to stationary phase and then platted on MPA in the form of suspension at different inoculation densities. The MPA with culture was MRET activated during the different periods of time (activation for 15 minutes, 30 minutes, 45 minutes, and 60 minutes respectively) following the requirements of sterility. Petri dishes with activated and non-activated medium (MPA with culture) were covered with glass caps (aerobic environment) and placed in the thermostat for cultivation at a temperature of 37oC during 18-24 hours.

After the cultivation the morphological and tinctorial properties of cultures were observed and the numbers of colonies grown on MPA were counted. The bacteriostatic activity of MRET activated nutrient medium (MPA) was measured as an Index of Bacteriostatic Activity (IBA). An Index of Bacteriostatic Activity is defined as a coefficient of the inhibition of growth and reproduction of pathogens in bacteriostatic medium, particularly in MRET activated nutrient medium. It is calculated as reduction of the number of colonies (CFU – Colony Forming Units) in MRET activated medium related to the control samples not exposed to the activation:

In order to verify the sterility of experiments Petri dishes with nutrient medium (MPA) without staphylococcal culture were exposed to the process of activation and then were kept in the thermostat. No colonies of culture were observed that confirms the sterility of environment.

RESULTS:

Following the investigation the direct correlation between times of activation (tact), initial concentrations of staphylococcal culture (N0) and a number of colonies grown on MRET activated medium were observed. The results are presented below in the form of a series of photos of Petri dishes with the colonies grown on MPA surfaces and the following diagrams based on the data of these experiments (Fig 7 – 12).

In the process of investigation the effect of MRET activation on the growth of staphylococcal culture at rather small initial concentration of pathogens was analyzed. The data corresponding to higher initial concentrations N0 > 103 bacteria/ml were not analyzed due to the difficulties related to calculation of very high values of a number of colonies, despite the fact of the high bacteriostatic activity of MRET activated nutrient medium in case of high initial concentrations.

Fig 7: The effect of time duration of MRET activation on the inhibition of growth of culture of Staphylococcus aureus Wood-46 with initial concentration of culture N0 = 103 bacteria/ml.

Fig 8: The effect of time duration of MRET activation on the inhibition of growth of culture of Staphylococcus aureus Wood-46 with initial concentration of culture N0 = 103 bacteria/ml. IBA – Index of Bacteriostatic Activity (reduction of the number of colonies related to the control samples not exposed to activation).

N0 = 102 bacteria/ml, Control

N0 = 102 bacteria/ml, tact = 15 min

N0 = 102 bacteria/ml, tact = 30 min

N0 = 102 bacteria/ml, tact = 45 min

N0 = 102 bacteria/ml, tact = 60 min

+Fig 9: The effect of time duration of MRET activation on the inhibition of growth of culture of Staphylococcus aureus Wood-46 with initial concentration of culture N0 = 102 bacteria/ml.

Fig 10: The effect of time duration of MRET activation on the inhibition of growth of culture of Staphylococcus aureus Wood-46 with initial concentration of culture N0 = 102 bacteria/ml. IBA – Index of Bacteriostatic Activity (reduction of the number of colonies related to the control samples not exposed to activation).

N0 = 10 bacteria/ml, Control

N0 = 10 bacteria/ml, tact = 15 min

Fig 11: The effect of time duration of MRET activation on the inhibition of growth of culture of Staphylococcus aureus Wood-46 with initial concentration of culture N0 = 10 bacteria/ml.

Fig 12: The effect of time duration of MRET activation on the inhibition of growth of culture of Staphylococcus aureus Wood-46 with initial concentration of culture N0 = 10 bacteria/ml. IBA – Index of Bacteriostatic Activity (reduction of the number of colonies related to the control samples not exposed to activation).

CONCLUSIONS:

1. MRET activation of the water based nutrient medium with suspended staphylococcal culture leads to the origination of high bacteriostatic activity of nutrient medium which depends on the time duration of activation and initial concentration of culture cells.

2. The bacteriostatic activity increases following the increase of time of activation (the times of activation up to 60 minutes were studied).

3. The efficacy of bacteriostatic activity increases following the decrease of initial concentration of the suspension of staphylococcal culture. The process of MRET activation is most effective on culture suspensions with the concentration not more than 103 bacteria/ml.

4. The results of investigation provide the evidence regarding the high efficacy of MRET activation on the inhibition of growth of colonies and reproduction of staphylococcal microorganisms in vitro. These results allow admitting that the process of MRET activation and the sterilization effect of MRET water can be applied in food industry and for water purification.

In this particular case, the blood sample of the subject before Activated Water ingestion shows free radical attack on cells. Note the “spiky” blood cells that take almost 90% of specimen. 30 minutes later, after Activated Water ingestion, the blood specimen shows non free radical environment.

In this particular case, the blood sample of the subject before Activated Water ingestion shows so called Rouleau formation when blood cells are stacked, forming a worm-like pattern. 30 minutes later, after Activated Water ingestion, most of the worm-like patterns broke up, forming individual patterns of nicely round spheres.

The examinations of influence of MRET activated water on plants was conducted during 3 months period at Kiev Institute of Plants Genetics of Ukrainian Academy of Sciences under supervision of Prof. V. Vysotskii, Kiev National University. MRET activated water accelerated the process of seed germination of several plants (particularly of cabbage, pumpkin, string bean, garden radish and peas) and enhanced their growth cycle.